A bioreactor system and method for producing a biopol ymer

ABSTRACT

Provided is a bioreactor system and a fermentation process employing continuous inline medium dilution in which concentrated medium and nutrients are blended with water or buffer and fed to the cell culture vessel of a bioreactor system, e.g. for production of antibodies and other recombinant proteins by mammalian cells.

FIELD OF THE INVENTION

The present invention relates to a bioreactor system and a method forproducing cells or a biopolymer using the bioreactor system. The methodsof the present invention are suitable for use in a manufacturing processfor preparing a polypeptide, in particular for preparing an activepharmaceutical ingredient for a pharmaceutical product.

BACKGROUND OF THE INVENTION

Traditionally, bacterial, yeast and mammalian cells for e.g. proteinproduction are primarily cultured as suspension cultures in bioreactors,also called fermenters. In such bioreactors the environmental conditionscan be precisely controlled by manipulating the supply of nutrients tothe cells and the removal of waste materials, and a stirring means maystir the culture medium within the reactor to provide for a homogeneousdistribution of the cells.

The bioreactor may be operated as a closed system in a batch orfed-batch process or as a continuous system in a so-called chemostat orperfusion process.

In a batch operation the culture medium usually contains a medium withthe necessary nutrients, for example glucose, vitamins, amino acids andminerals. During fermentation, these are consumed so that the mediumbecomes more and more deprived in nutrients. At the same time, theconcentration of waste products increases, which ultimately results ininhibition of cell growth. In a fed-batch process one or more of thenutrients are fed (supplied) to the bioreactor during cultivation toachieve better growth conditions and higher cell densities.

In a continuous system such as a chemostat fresh medium is continuouslyadded, while culture liquid is continuously removed to keep the culturevolume constant. By changing the rate at which medium is added to thebioreactor, the growth rate of the microorganism cells can becontrolled. For cells with a high growth rate such as yeast and bacteriacells, the chemostat typically removes cells from the medium along withthe culture liquid in order to maintain a desired cell density.

A perfusion process is a special type of continuous process in which asuspension cell culture is continuously supplied with fresh medium tothe bioreactor while spent culture media is continuously harvested. Thecells are continuously filtered or otherwise separated from the harveststream and returned to the bioreactor to maintain a uniform celldensity. The constant addition of fresh medium and elimination of wasteproducts provides the cells with the optimal environment to achieve highcell concentrations and thus higher productivity. This allows prolonginghealthy cultures, potentially at high cell density, as well as a shortresidence time of the product in the bioreactor. This is more favourablefor product quality and is required for the production of unstablepolypeptides. Another advantage of the perfusion mode is that it allowsthe use of smaller bioreactors compared with fed-batch processes, whichprovides benefits such as reduced clean-in-place operation and thepossibility to use disposable bioreactors instead of stainless steelreactors due to the smaller working volumes. Moreover, product may becontinuously harvested by taking out medium (with cells and product) orvia a so-called bleed.

Due to an increasing demand for biologically produced medicinal productssuch a complex polypeptides, including antibodies and other recombinantproteins, perfusion processes are becoming a much more common productionplatform due to the high productivity in relation to the size of thebioreactor.

However, in large-scale continuous processes, media and harvestlogistics require specific attention. Depending on the scale of thebioreactor and the chosen perfusion rate, it may be necessary to provideseveral bioreactor volumes of fresh medium daily to the bioreactor, e.g.500 L to 6000 L or more, and to collect a similar daily volume fordownstream purification. Thus, huge amounts of fresh medium have to beprepared daily for large-scale production facilities, for example up toabout 7500 L for a 2500 L reactor, which clearly involvespractical/logistical as well as economic challenges. Since purchase andhandling of the cell culture medium are some of the most expensiveaspects of the production of mammalian cell products, there is an acuteneed for systems that can optimize medium use and handling forindustrial large-scale bioreactor systems.

The present invention addresses the need for improved and more efficientutilization and handling of cell culture media in large-scale bioreactorsystems, in particular in continuous fermentation processes, and forimproving productivity in such bioreactor systems.

SUMMARY OF THE INVENTION

The present invention provides a bioreactor system and a fermentationprocess employing continuous inline medium dilution in whichconcentrated medium and nutrients are blended with water or buffer andfed to the cell culture vessel of a bioreactor system. This systemprovides several advantages, one advantage being that medium andnutrients can be mixed from concentrated solutions with water or bufferby inline dilution immediately prior to use, thereby significantlyreducing the container size requirement and making the overall processmore efficient. Another advantage is that different media components maybe blended from independent stock solutions having differentconcentrations and/or which are kept at different temperatures. Furtheradvantages will be apparent from the disclosure below.

One aspect of the invention relates to a bioreactor system for producinga product selected from a cell and a biopolymer expressed by a cell, andwherein the bioreactor system comprises:

-   -   a cell culture vessel (5) comprising a product harvest module        (6);    -   optionally, a bleed outlet (9);    -   a medium container (1;2);    -   a water and/or buffer supply (3); and    -   an inline medium dilution system (4) for diluting concentrated        medium from the medium container, the inline medium dilution        system having an inlet and an outlet, wherein the inlet is in        fluid communication with the medium container and the        water/buffer supply, and wherein the outlet is in fluid        communication with the culture vessel.

Another aspect of the invention relates to a method for producing abiopolymer using the bioreactor system disclosed herein, the methodcomprising:

-   -   (a) fermenting cells expressing the biopolymer in a suitable        medium under suitable conditions to allow expression of the        biopolymer by the cells,    -   (b) harvesting the biopolymer by removing medium comprising        biopolymer and impurities via the product harvest module, and    -   (c) isolating the biopolymer from the harvested medium;    -   wherein, during fermentation, the method further comprises:    -   adding concentrated medium from a medium container and water or        buffer from the water/buffer supply to an inlet of the inline        medium dilution system to result in diluted medium, and feeding        the diluted medium containing nutrients to the culture vessel        through an outlet to replenish nutrients consumed by the cells        and to compensate for medium removed for harvesting of the        biopolymer.

In a further aspect, the invention relates to the use of a bioreactorsystem as described herein for producing a product selected from a celland a biopolymer expressed by a cell.

Further objects of the present invention will become apparent in view ofthe present description, figures and claims.

DRAWING DESCRIPTION

FIG. 1 is a schematic illustration of a basic bioreactor system of theinvention.

FIG. 2 is a schematic illustration of an alternative embodiment of thebioreactor system of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The bioreactor system of the present invention includes several basiccomponents, including a cell culture vessel comprising a product harvestmodule, at least one medium container, a water and/or buffer supply, aninline medium dilution system, and optionally a bleed outlet. It mayalso contain additional components such as an impurity filter unit. Thecharacteristics of the individual components of the system, and theirfunction in the context of the method of the invention for producing abiopolymer will be explained in detail in the following.

Bioreactor

As used herein the term “bioreactor” refers to any device or system thatsupports a biologically active environment, for example for cultivationof cells for production of a biological product. Bioreactors may rangein size from a few liters to several cubic meters (i.e. several 1000liters), and may be a conventional bioreactor based on a culture vesselof e.g. stainless steel or glass or a “single-use” bioreactor based on adisposable material such as a disposable bag.

While bioreactors have in the past typically been of the conventionaltype, most often based on stainless steel tanks, disposable bioreactorsbased on a disposable bag, typically made of a multilayer plasticmaterial, are becoming more prevalent. For agitation, some single-usebioreactors use stirrers similar to those of conventional bioreactors,but with stirrers integrated into the plastic bag, while othersingle-use bioreactors are agitated by means of a rocking motion.Stirred single-use bioreactors may have a volume of up to severalthousand liters, e.g. 2000 to 5000 liters, while rocking single-usebioreactors typically have a volume of up to about 1000 liters.

Single-use bioreactors have several advantages compared to conventionalbioreactors, including reduced cleaning and sterilization demands, alongwith significant accompanying cost savings. In addition, complexqualification and validation procedures for pharmaceutical productioncan be simplified, and there is a reduced risk of cross contamination.Further, since single-use bioreactors contain fewer parts compared withconventional bioreactors, initial and maintenance costs are reduced.

Based on the mode of operation, a bioreactor may be classified as batch,fed-batch or continuous. Examples of continuous bioreactors are achemostat and a perfusion bioreactor. The bioreactor is typicallyequipped with one or more inlets for supplying culture medium to thecells, and with one or more outlets for harvesting product or emptyingthe bioreactor. Additionally, the bioreactor may be equipped with atleast one outlet constructed in such a way that a separation device canbe attached to the bioreactor. Typically, the bioreactor's environmentalconditions such as gas (i.e., air, oxygen, nitrogen, carbon dioxide)flow rates, temperature, pH and dissolved oxygen levels, and agitationspeed/circulation rate can be closely monitored and controlled.

The bioreactor may optionally also include a separate inlet for addingcomponents such as vitamins or growth factors. In this case, suchcomponents may be added to the cell culture vessel in addition to thediluted medium, and may be either in concentrated or diluted form.

In a preferred embodiment, the bioreactor system of the invention is acontinuous system, i.e. a perfusion or chemostat bioreactor. Perfusionbioreactors are typically used for cultivation of mammalian cells, whilechemostat bioreactors are typically used for cultivation ofmicroorganisms such as bacteria or yeast cells.

In a other embodiment the bioreactor system is a continuous productionsystem.

Cell Culture Vessel

A “cell culture vessel” as used herein refers to an integral part of abioreactor system in which cells are grown under suitable conditions ina suitable medium. The cell culture vessel may be a single-use vessel,e.g. a disposable bag, or a conventional reusable vessel, typically astainless steel or glass vessel, as explained above. Stainless steelvessels are typically configured with predefined port assemblies,whereas single-use bags use pre-sterilized plastic cultivation chambersthat are discarded after use. This eliminates space-consuming andexpensive clean-in-place (CIP) and steam-in-place (SIP) installationswhile reducing production turnaround times.

The cell culture vessel of the invention typically has a volume of atleast 50 L, preferably at least 100 L, more preferably at least 250 L,and still more preferably at least 500 L. In many cases, the volume willbe still higher, e.g. at least 1000 L or at least 2000 L.

Medium Container

A “medium container” as used herein refers to any kind of container,e.g. a rigid tank of e.g. steel, glass or plastic or a collapsibleand/or disposable bag, that holds cell culture medium and/or nutrients.In the context of the present invention, the medium container isconnected to at least one inlet end of an inline dilution system, andthe cell culture medium and/or nutrients will typically be present inthe medium container in a more concentrated form than concentration ofthe same medium or nutrients when present inside the culture vessel.

In one embodiment of the invention, the bioreactor system may comprisetwo or more medium containers, each of which is in fluid communicationwith an inlet of the inline dilution system. The use of two or moremedium containers may be advantageous in order to be able to furtherreduce container size, space requirements, etc., for example by usingone container to hold medium components or nutrients having a relativelylow solubility and another container to hold other medium components ornutrients that have a higher solubility. By having the low solubilitycomponents in a separate container, the volume of the containercomprising the other components that have a higher solubility may bereduced. A further advantage of this approach is that components with alow solubility can be stored under conditions that contribute toincreasing their solubility, for example by means of suitable pHadjustment. Medium and nutrients from the two or more medium containerscan be mixed in suitable amounts via the inline medium dilution systemin order to obtain a desired final culture medium composition. This isdiscussed in more detail further below in connection with the inlinemedium dilution system, from which it will be apparent that each mediumcontainer may be in direct or indirect fluid communication with an inletof the inline medium dilution system.

In one embodiment at least one medium container in fluid communicationwith the inline dilution system has a volume of at least 10 L, such asat least 50 L, such as at least 100 L, e.g. at least 250 L.

In an embodiment, the concentrated medium in the medium container(s) maybe kept at a reduced temperature of e.g. 1-10° C., such as about 5° C.In this case, the diluted medium is preferably pre-heated prior to beingadded to the culture vessel. This may be performed by heating thediluted medium as such or by mixing the concentrated medium withwater/buffer that has been pre-heated to a desired temperature. Forexample, the diluted medium may be pre-heated to the same temperature asthe temperature of the medium inside the culture vessel.

Inline Medium Dilution System

Inline dilution refers to the concept of mixing a concentrated solutionand water (or some other diluent, e.g. an aqueous buffer in the presentcontext) inside a processing line to produce a normal strength,process-ready solution. Inline dilution systems (sometimes called“on-site blending systems”) also provide many advantages over purchasingpre-mixed and diluted cell culture media. By using a blending system, asingle container or medium concentrate produces many times its volume indiluted medium. Thus, a single volume of concentrated medium, used toproduce the equivalent of many volumes of dilute medium via the dilutionsystem, greatly reduces facility costs associated with fabrication oflarge tanks, reduces floor space requirements, and reduces validationand quality control costs as well as spoilage and disposal costs ofnon-compliant, out-of-date or unused blended solutions. Costs associatedwith medium delivery and handling are also greatly reduced. In addition,onsite dilution and mixing increases the variety of mediumconcentrations and mixtures that are immediately available, withoutrequiring a corresponding increase in the number of different types ofmedia and nutrient supplements that must be purchased, thereby reducingfacility and operating costs and providing the logistical andadministrative advantage of reduced inventory.

Another advantage of inline dilution is that if, for example, onecomponent of a medium is consumed at a faster rate at a high celldensity than at a low cell density, the medium can be compensated forthis by mixing in a higher concentration of this component.

Inline medium dilution of concentrated medium or nutrient solutions withwater or buffer must be made within tight specification ranges for pH,conductivity, osmolality, and temperature, which are critical processparameters. This requires that a precise mixture of the concentratedsolution and water can be delivered with minimal deviation over time.Also, the solution must be well mixed prior to delivery to the cellculture vessel.

The inline medium dilution system may, in its most basic form, be asimple system of tubes or pipes from which concentrated medium andwater/buffer, respectively, are supplied, and that connect with eachother at one end before being led into the inlet of the cell culturevessel. However, more typically the inline medium dilution system willbe a more advanced automated system that allows two or more liquidstreams to be brought together in a controlled fashion to meet a targetdiluted solution concentration. Inline dilution systems are commerciallyavailable from different suppliers such as from Novasep, GE Healthcareor for example the system IBD™ 1K Inline Buffer Dilution System fromAsahi Kasei Bioprocess (disclosed in U.S. Pat. No. 8,271,139). Suchsystems are capable of making multi-component blends of up to 20×concentrates and produce a ready-to-use solution offering total blendflow rates of more than 1000 L/h.

As used herein, the term “water and/or buffer supply” or “water/buffersupply” is intended to encompass any supply of water or buffer for usein diluting the concentrated medium. This can include containers thathold water or a pre-mixed buffer solution for mixing with theconcentrated medium as well as e.g. dilution systems in which aconcentrated buffer is mixed with water prior to use in the inlinemedium dilution system. Further, in the event the inline medium dilutionsystem dilutes the concentrated medium with water only, the water supplymay be any supply of suitable water, whether stored in a tank or othercontainer or supplied as needed in purified form using e.g.ultrafiltration or reverse osmosis.

In one embodiment of the present invention, the inline dilution systemhas a total blend flow rate of at least 1 L/min, such as at least 2L/min, such as at least 5 L/min, such as at least 10 L/min.

There are several approaches for operating the blend procedure. Forexample, some systems blend the final solution based on conductivityand/or pH data provided by conductivity and pH process analyzers,whereas other systems use volumetric flow rate as the primary means ofcontrol, since inline pH and conductivity meters have an inherenttendency to drift and improper calibration may result in false readings.

The inline medium dilution system e.g. may be constructed such that allthe media components and nutrients are pre-mixed into a singleconcentrated solution designed to be diluted with water or buffer bye.g. a factor of 1.5 or more, typically two or more, for example afactor of three, a factor of four or a factor of five, or even higher,such as factor of eight or ten, in the inline dilution system andsubsequently provided to the cell culture vessel. In this case, thesystem will use a single medium container.

Alternatively, the system may employ two or more medium containers, e.g.containing different medium components with different solubilities asdiscussed above. In this case, the system may be constructed such thatdifferent media components and nutrients having different concentrationsare led into a single mixing chamber by different inlets at differentflow rates and diluted in the mixing chamber with water or buffer to thedesired concentration (e.g. to the concentration of the medium in thecell culture vessel), whereafter the diluted mixture is provided to thecell culture vessel. Another option in the case of multiple mediumcontainers is for each medium container to be connected to a separatemixing chamber for dilution with water or buffer. The separate mixingchambers can be further connected to a common mixing chamber, whereindiluted medium from two or more individual separate mixing chambers ismixed together before being led into the culture vessel via a singleinlet, or alternatively, diluted medium from individual separate mixingchambers may be led into the culture vessel by way of multiple inlets,e.g. one inlet for each mixing chamber.

In one embodiment of the invention, the inline medium dilution systemmay be connected to a sensor located within or outside the cell culturevessel that can measure the concentration or the amount of medium or ofselected components or nutrients in the cell culture vessel. The inlinedilution system may in this case be operated as an automated system,allowing the concentration or the amount of medium or selected mediumcomponents or nutrients in the cell culture vessel to e.g. be keptconstant in the event the perfusion rate is changed or a bleed is madeto decrease the cell density in the cell culture vessel, or to otherwisebe regulated as desired.

The inline medium dilution system may have inline monitoring and controlof the dilution process using instrumentation such as mass flow metersand/or analytical instruments such as pH, conductivity or near-infrared(NIR) instrumentation. A programmable logic controller may integrate theoperation and control of all components in the system.

If desired, a holding step can be used following mixing of theconcentrated medium with the water/buffer to produce the diluted medium.The system of the invention may therefore optionally include a “holdingcontainer”, e.g. a holding tank, between the inline medium dilutionsystem and the cell culture vessel, i.e. such that outlet of the inlinemedium dilution system is in indirect fluid communication with the cellculture vessel. The holding tank/container may function not only totemporarily hold the diluted medium, but may also, if desired, beadapted to provide additional mixing of the diluted medium before it isled into the cell culture vessel. This may e.g. be advantageous whenusing multiple medium containers with e.g. one container for the lowsolubility components.

Product Harvest Module

In one embodiment the product harvest module is in its most simple formjust an outlet leading to a container or bag suitable for collecting theproduct along with cells, impurities and medium for storage or furtherdownstream processing. It may also be a separation device capable of,for example, separating polypeptides from cells, cell debris andimpurities larger than the product of interest. The product harvestmodule may be operated to continuously harvest the product in a harveststream that is collected for further downstream processing.

The product harvest module may also be a separation device such as acell retention device that can separate cells from the product harveststream such that the cells are retained in the cell culture vessel.

There are two major classes of techniques for the separation of cellsfrom the medium, namely by gravitational or centrifugal sedimentation,or by filtration (for example tangential filtration such as alternatingtangential-flow filters, e.g. axial rotation filtration or as spinfilters, flow filters, vortex filters or cross flow filtration). In oneembodiment, the product harvest module is a separation device based ongravitational or centrifugal sedimentation. In a preferred embodimentthe product harvest module is a separation device based on alternatingtangential-flow filtration.

Gravitational separation is an industrial method of separating twocomponents, either a suspension or a dry granular mixture in whichseparation of the components by gravity is practical. This method can beused to separate out solids from a liquid mixture if the solids are notsoluble in the liquid. The skilled person will know how to attachsuitable gravitational separation devices to a bioreactor.

Centrifugal separation is another well-known technique to separate outparticles in suspension. Commercially available separators utilizingcentrifugal force for separation fall in one of two categories, rotarycentrifuges or hydrocyclones. Hydrocyclones are operated by creating aphysical vortex within a cylindrical vessel, generating centrifugalforce. The heavier phase is forced to the outside portion of the fluidand the lighter fluid stays in the inside as a core. As the fluidcontinues flowing, the separated portions are directed to differentoutlets. Suitable centrifugal separation devices are known andcommercially available, and use of these together with a bioreactor willbe familiar to those skilled in the art.

In another embodiment the product harvest module is a filter unit, inwhich case the product harvest module may be referred to as a productfilter. A product filter is often selected with a pore size in the rangeof from a nominal molecular weight cutoff (NMWC) of about 50,000 daltons(50 kDa) to about 2 μm, such as from an NMWC of about 100,000 daltons(100 kDa) to a pore size of about 1 μm.

As known to the skilled person, a suitable product filter cut-off willdepend on the size of product of interest. In a preferred embodiment,the product filter has an NMWC pore size of at least about 1.5 times theMW of the biopolymer (e.g. polypeptide) of interest. For instance, ifthe MW of a polypeptide of interest is 100,000 (100 kDa) the preferredcut-off of the product filter will be an NMWC of at least 150,000 (150kDa). More preferably, the product filter has an NMWC pore size of atleast 2 times the MW of the polypeptide of interest.

When the cells present in the bioreactor reach a satisfactory celldensity or when there is sufficient product present in the outflowthrough the harvesting outlet, harvest of the product may be initiated.This may be determined by measuring the cell density, for example usinga spectrophotometer, or by measuring the amount of the product ofinterest by known means, for example using a suitable protein assaymethod in the case of a polypeptide product.

Impurity Filter Unit

Numerous specialized filters and filtration methods have been developedto separate materials according to their chemical and physicalproperties. Known filters include flat surface filters, pleated filters,multi-unit cassettes, and tubular forms such as hollow fibers. For theinvention described herein any system of ultrafiltration technology canbe applied as long as sterility can be ensured.

As used herein the term “impurities” refers to undesired chemical orbiological compounds produced by the cells present in the bioreactor, orwhich arise from cells that die or break open during the fermentationprocess. Impurities include e.g. ethyl alcohol, butyl alcohol, lacticacid, acetone ethanol, gaseous compounds, peptides, lipids, ammonia,aromatic compounds, and DNA and RNA fragments, as well as mediacomponents.

Examples of filtration systems applicable for use in the production ofpolypeptides and removal of impurities as described herein are systemssuch as cartridge systems, plate and frame systems, and hollow fibersystems. The systems can be operated by pumping liquid over themembrane, by vibration (e.g. as supplied by PallSep™) or by alternatingtangential flow (ATF), and both polymeric and ceramic membranes are wellsuited for the filtration process. A skilled person will be able toselect a membrane with suitable properties.

Hollow fiber membranes have been successfully employed in a wide varietyof industries, and have several benefits that include high membranepacking densities the ability to withstand permeate back-pressure, thusallowing flexibility in system design and operation. Hollow fibercartridges can operate from the inside to the outside during filtration,allowing process fluid (retentate) to flow through the center of thehollow fiber and permeate to pass through the fiber wall to the outsideof the membrane fiber. Tangential flow can help limit membrane fouling.Other operating techniques that can be employed with hollow fibermembrane systems include back flushing with permeate and retentatereverse flow.

Accordingly, the filter may be located in an external filter moduleattached to the bioreactor. Alternatively, the impurity filter may belocated inside the bioreactor. The filter unit can also contain pumps orsystems for preventing fouling of the filter such as an ATF system orthe PallSep™ system in which controlled horizontal oscillation moves themembrane elements through the feed fluid. The oscillation generatesvibrational energy at the membrane surface, giving shear (higher thanthat typically generated in conventional tangential flow filtrationsystems) that is limited to a small boundary layer above the membranesurface, and which is not applied to the bulk of the fluid. This ensuresthat even in high solids feed streams, the membranes do not cake withthe retained species.

The system can, depending on the metabolites to be removed and theproduct in question, be equipped with membranes with a molecular weightcut-off value from a few hundred to tens of thousands. Often membraneswith a cut-off between 1000 and 20,000 (1-20 kDa) are used. The benefitof using a membrane with a cut-off of about 10,000 (10 kDa) or below,preferably around 5000 (5 kDa), is that growth factors such as insulinand IGF-1 will be retained in the bioreactor.

During an extended run, it is possible to change the filters andresterilize the system without terminating the fermentation.

The skilled person will be able to select a suitable filter type forremoval of impurities and a suitable membrane nominal molecular weightcutoff (NMWC) pore size with respect to allowing impurities to perfuseout of the impurity filter and harvest the polypeptide of interestthrough the product harvesting outlet.

In one embodiment, the impurity filter unit is selected from a membranefilter, a gravitational separation unit and a centrifugal separationunit.

The impurity filter is often selected with an NMWC within the range of1000 to 30,000 (1-30 kDa), such as in the range of 2000 to 20,000 (2-20kDa) or in the range of 2000 to 15,000 (2-15 kDa). However, if theproduct is a cell an impurity filter may be selected with an NMWC in therange of 1000 to 500,000 (1-500 kDa), but normally it is preferred thatthe impurity filter has a cutoff of less than 20,000 (20 kDa). Thus, inone embodiment the impurity filter unit is a membrane filter having anNMWC pore size of at least 1000, such as within the range of 2000 to15,000.

In a preferred embodiment the impurity filter unit is a membrane filterhaving a molecular weight cut-off (NMWC) pore size of a maximum of 80%of the molecular weight (MW) of the product (e.g. polypeptide) ofinterest. For instance if the MW of the polypeptide of interest is100,000 (100 kDa) the preferred maximum cut-off of the impurity filterwill in this case be 80,000 (80 kDa). More preferably, the impurityfilter has an NMWC pore size of a maximum of 50% of the MW of thepolypeptide of interest. Thus, in one embodiment the impurity filter hasa molecular weight cut-off (NMWC) pore size of a maximum of 80% of theMW of the biopolymer, such as a maximum of 50%.

Bleed Outlet

The “bleed outlet” is an outlet from the cell culture vessel that allowsmedium containing cells, cell debris and impurities to be removed fromthe cell culture vessel. It may be constructed as separate outlet, or itmay be built together with the product harvest module. Bleeding of cellshelps to ensure optimal productivity in continuous fermentationprocesses, in particular for perfusion processes, as it serves to e.g.improve overall cell culture viability, to avoid accumulation of deadcells and to prevent filter clogging. For cultures operated in perfusionmode it is a common practice to make bleeds daily if the viability ofthe cells drops to for example below 80% or when the cell densityreaches a certain level, for example around 30 million cells/ml. Duringthe bleeds up to 10% of the medium in the cell culture vessel maytypically be removed, and this amount may increase with increasing celldensity such that the bleeds may be used to regulate the cell density inthe cell culture vessel.

While perfusion bioreactors will normally contain a bleed outlet,chemostat bioreactors used for e.g. bacteria or yeast cultivationgenerally do not include a bleed outlet.

Cell Culture Medium

As used herein “medium” refers to a cell culture medium. Numerous cellculture media are known and commercially available. Such media typicallyhave a composition which is adapted for cultivation of certain types ofcells and may comprise salts, amino acids, vitamins, lipids, detergents,buffers, growth factors, hormones, cytokines, trace elements andcarbohydrates. Examples of salts include magnesium salts, for exampleMgCl₂×6H₂O, and iron salts, for example FeSO₄×7H₂O, potassium salts, forexample KH₂PO₄, KCI, sodium salts, for example NaH₂PO₄ or Na₂HPO₄, andcalcium salts, for example CaCl₂×2H₂O. Examples of amino acids are the20 naturally occurring amino acids, for example histidine, glutamine,threonine, serine, methionine. Examples of vitamins include ascorbate,biotin, choline, myo-inositol, D-panthothenate and riboflavin. Examplesof lipids include fatty acids, for example linoleic acid and oleic acid.Examples of detergents include Tween® 80 and Pluronic® F68. An exampleof a buffer is HEPES. Examples of growth factors/hormones/cytokinesinclude IGF, hydrocortisone and (recombinant) insulin. Examples of traceelements include Zn, Mg and Se. Examples of carbohydrates includeglucose, fructose, galactose and pyruvate. Examples of other componentsthat may be included in the medium are soy peptone and ethanol amine.The skilled person will be familiar with suitable media and mediasupplements as well as suitable conditions with respect to specificexpression cells and polypeptides of interest.

Silicon-based antifoams and defoamers or nonionic surfactants such ascoblock polymers of ethylene oxide/propylene oxide monomers may be addedto the medium during fermentation.

The pH, temperature, dissolved oxygen concentration and osmolarity ofthe cell culture medium will depend on the particular type of cell, andwill be chosen such that they are optimal for the growth andproductivity of the cells in question. The person skilled in the artwill know how to determine the optimal conditions such as pH,temperature, dissolved oxygen concentration and osmolarity for a givenculture. Usually, the optimal pH for mammalian cells is between 6.6 and7.6, the optimal temperature is between 30 and 39° C., and the optimalosmolarity is between 260 and 400 mOsm/kg. For microbial systems the pHmay be between 3 and 8 and the temperature from 20 to 45° C.

The solubility of the different medium components varies considerably,as many of the components will have a high solubility and thus be easilydissolved in water whereas other components such as certain vitamins,amino acids, lipids and growth factors have a low solubility in water.For this reason, cell culture media are normally prepared by mixingtogether all the components as a ready-to-use composition.

In one embodiment of the present invention the medium is made such thatcomponents that are easily dissolved in water are prepared together inone lot, and components with a low solubility and that are difficult todissolve in water are prepared together in another lot. The two (ormore) lots are then separately dissolved in water so as to produce two(or more) concentrated media fractions having desired concentrations ofthe individual components. The concentrated media fractions may forexample be prepared as solutions wherein the media components are 2times, 3 times, 4 times, or 5 times or more, e.g. up to 10 times, asconcentrated as the components in the culture vessel.

Cells

As used herein the term “cell” can include both prokaryotic andeukaryotic cells.

Expression of biopolymers, in particular polypeptides, for therapeuticuse has been accomplished using bacteria, yeast and mammalian cells, andthe skilled person will be familiar with numerous suitable expressioncells for production of a given product. The cells expressing thebiopolymer (e.g. polypeptide) may thus be selected e.g. from the groupconsisting of E. coli, Bacillus, yeast of the genus of Saccharomyces,Pichia, Aspergillus, Fusarium, or Kluyveromyces, CHO (Chinese hamsterovary) cells, hybridomas, BHK (baby hamster kidney) cells, myelomacells, HEK-293 cells, PER.C6® cells, human lymphoblastoid cells andmouse cells, for example NSO cells.

In the context of the present invention, the cells are preferablyeukaryotic cells, in particular mammalian cells. Preferred cell linestypically employed for mammalian cell culture include CHO cells, NSOcells, BHK cells, HEK-293 cells and PER.C6® cells. In one embodiment,the cell is a CHO cell such as a CHO DG44 cell, for example undercontrol of Chinese hamster EF-1α regulatory sequences.

Since the invention as described preferably operates using a high celldensity, one may advantageously use a cell culture medium with a highcell density from one fermentation to re-start (i.e. seed) a newfermentation. A high viable cell density in this context is typically adensity of at least 10 million cells/ml, preferably at least 20 millioncells/ml, more preferably at least 30 million cells/ml, e.g. at least 40million cells/ml, such as at least 50 million cells/ml. A preferred celldensity is from at least 10 million cells/ml to 100 million cells/mlsuch as from 10 million cells/ml to 80 million cells/ml.

In some cases it may be convenient to grow cells to a desired celldensity in one bioreactor and then transfer the cells to a secondbioreactor for inducing the expression of the polypeptide by adding aninducer (for cells that are under control of an inducible promoter) orby changing the temperature and/or the pH of the medium. In such caseimpurities may also be removed via the separation device of the firstbioreactor using a desired flow rate and via the separation device ofthe second bioreactor using the same or a different desired flow rate.

Biopolymers

The term “biopolymer” as used herein means a polypeptide, protein,nucleic acid or virus particle, which can be native or biologically orsynthetically modified, including fragments, multimers, aggregates,conjugates, fusion products etc. In one embodiment, the biopolymer is arecombinant protein such as an antibody. In another embodiment, thebiopolymer is a virus particle or part thereof, for example a proteincoat, for use as a vaccine. In a further embodiment, the biopolymer maybe a nucleic acid.

In a preferred embodiment of the present invention the product is apolypeptide or protein. As used herein, the terms “protein” or“polypeptide” may be used interchangeably and refer to any chain ofamino acids, regardless of length or post-translational modification.Proteins can exist as monomers or multimers, comprising two or moreassembled polypeptide chains, fragments of proteins, polypeptides,oligopeptides, or peptides.

Examples of polypeptides of interest that may be produced using thesystems and methods of the invention include recombinant therapeuticproteins such as antibodies or fragments thereof, blood clottingfactors, cytokines, enzymes, peptide hormones, etc. Specific examples ofsuch proteins include human growth hormone, follicle-stimulatinghormone, Factor VIII, Factor VII, Factor IX, erythropoietin (EPO),granulocyte colony-stimulating factor (G-CSF), alpha-galactosidase A,alpha-L-iduronidase (rhIDU; laronidase),N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase), DNAse, tissueplasminogen activator (TPA), glucocerebrosidase, interferons (IF) suchas interferon-alpha, interferon-beta and interferon-gamma, insulin,insulin derivatives, insulin-like growth factor 1 (IGF-1), tenecteplase,antihemophilic factor, human coagulation factor, and etanercept; andantibodies such as Trastuzumab, Infliximab, Basiliximab, Belimumab,Daclizumab, Adalimumab, Abciximab, Afutuzumab, Alemtuzumab, Cetuximab,Daclizumab, Denosumab, Eculizumab, Edrecolomab, Golimumab, Ibritumomabtiuxetan, Mepolizumab, Motavizumab, Natalizumab, Ofatumumab, Omalizumab,Oregovomab, Palivizumab, Pemtumomab, Pertuzumab, Ranibizumab, Rituximab,Tefibazumab and Zanolimumab.

In a particular embodiment of the present invention the product is anantibody or a fragment thereof, where a fragment can e.g. be a Fabfragment, Fv fragment or single chain Fv (scFv) fragment.

Polypeptides are expressed under the control of regulatory sequencescalled promoter sequences. Cells expressing a polypeptide may be underthe control of a constitutive promoter (i.e. unregulated sequences; thisallows for continual transcription of the associated gene) or undercontrol of an inducible promoter (regulatory sequences induced by thepresence or absence of biotic or abiotic factors). In some cases, if thepolypeptide of interest has limited stability or exhibits toxic effectson the host cell, it may be convenient to express it under control of aninducible promoter such that the cells first are grown to a desired celldensity, after which expression of the polypeptide is induced by addingan inducer or by changing the temperature and or the pH of medium. Anexample of a constitutive promoter is a Chinese hamster EF-1α promoter.In one embodiment, the biopolymer is expressed under control of Chinesehamster EF-1α regulatory sequences.

By use of the system and method of the invention, it is possible toexpress polypeptides such as antibodies with high productivity. Thus, inone embodiment, the cells express a polypeptide, e.g. an antibody, andhave a productivity of at least 1 gram/L/day, and preferably higher,such as 2 or 3 gram/L/day or more.

The isolated product (e.g. polypeptide) of interest produced using thesystem and method of the invention will be purified by methods known inthe art for the given product, formulated into a final commerciallyrelevant composition of interest (e.g. a pharmaceutical composition),and packaged in a suitable container.

Fermentation Process

As explained elsewhere herein, the system of the invention is preferablya continuous system, i.e. the fermentation is performed as continuousfermentation. In a preferred embodiment, the product produced by themethod is a polypeptide, and the fermentation is performed as aperfusion process, i.e. a process in which a suspension cell culture ina bioreactor is continuously supplied with fresh medium while spentculture medium is continuously harvested, with cells being continuouslyfiltered from the harvest stream and returned to the bioreactor tomaintain a uniform cell density.

Except as otherwise described herein, the perfusion process may beperformed as generally known in the art. A typical process may thusinvolve, following inoculation of the bioreactor, e.g. about 2-3 days inwhich the cells are grown without perfusion in order to obtain aninitial desired cell density, followed by initiation of perfusion (i.e.harvest) at a low level of e.g. about 0.5-1 reactor volume per day,after which the perfusion rate is increased to e.g. about 1.5-3 reactorvolumes per day once the cell density has increased further. The term“reactor volume” in this context will be understood as corresponding tothe working cell culture vessel volume of the particular system. Theprocess is continuously monitored as known in the art and as otherwiseexplained herein, such that growth conditions, medium concentration,cell density, pH etc. are maintained within desired specifications.

The level of the harvest stream for a given perfusion process, i.e. thelevel used for the majority of the fermentation, will be able to bedetermined by the skilled person taking into consideration thecharacteristics of the individual bioreactor system and process, butwill often be in the range of from about 0.5 to about 3 reactor volumesper day, such as from about 1 to about 3 reactor volumes per day, e.g.from about 1.5 to about 2.5 reactor volumes per day. The perfusionprocess is often performed for about 3-6 weeks, but may last evenlonger, such as up to about 2 months or more.

Persons skilled in the art will be aware that the temperature of themedium in the cell culture vessel is a key factor for productivity ofthe cells, with a temperature in the range of about 30-38° C. oftenbeing optimal, and that it may be advantageous to employ a temperaturereduction during the fermentation. Such procedures are well-known, inparticular for mammalian cells such as CHO cells, and typically involvean initial fermentation phase at a first temperature of e.g. about 37°in order to obtain a desired cell density, followed by a reduction intemperature to, for example, about 32-35° for the remainder of thefermentation in order to increase expression of the polypeptide productand reduce cell division.

Detailed Drawing Description

The following non-limiting drawing descriptions are for example purposesonly.

A bioreactor system of the invention in its basic form is illustratedschematically in FIG. 1, which shows a medium container (1) and awater/buffer container (3), both of which are in fluid communicationwith an inline medium dilution system (4). Concentrated medium from themedium container (1) is mixed in the inline medium dilution system (4)with water or buffer from the water/buffer container (3) in appropriateamounts to obtain a desired dilution of the concentrated medium. Dilutedmedium is then fed as needed to the cell culture vessel (5), which iswhere fermentation of the cells takes place in order to produce e.g. apolypeptide or other biopolymer. Water or buffer may in additionoptionally be led directly from the water/buffer container (3) to thecell culture vessel (5) (illustrated by the dashed line between thetwo).

Connected to the cell culture vessel (5) is a product harvest module (6)for removing the biopolymer product along with cells, impurities andmedium. The removed material may be led to a storage vessel (7) fortemporary storage prior to being purified in the downstream processing(10), or it may be led directly from the harvest module (6) to thedownstream processing (10). In a perfusion system, cells that areremoved from the cell culture vessel (5) via the product harvest module(6) are typically returned to the cell culture vessel (5) (dashed linefrom (6) to (5)).

Connected to the cell culture vessel (5) is optionally also an impurityfilter unit (8) for separating out undesired purities, and a optionallya separate bleed outlet (9) that allows medium containing cells, celldebris and impurities to be removed from the cell culture vessel (5). Asexplained above, the bleed outlet (9) may either be constructed as aseparate unit or it may be built together as a single unit with theproduct harvest module (6).

FIG. 2 shows an alternative embodiment of the bioreactor systemillustrated in FIG. 1. In FIG. 2 there are two medium containers (1) and(2), for example where one medium container is for relatively highlysoluble medium components and the other medium container is for poorlysoluble medium components. The system can also include one or moreadditional medium containers (not shown) as desired. Concentrated mediumfrom the two medium containers (1) and (2) enters the inline mediumdilution system (4), where it is mixed with water or buffer from thewater/buffer container (3).

FIG. 2 further shows an optional holding tank (11) inserted between theinline medium dilution system (4) and the cell culture vessel (5). Theholding tank (11) may be used as necessary for temporarily holdingdiluted medium from the inline medium dilution system (4), andoptionally to provide additional mixing of the diluted medium before itis led to the cell culture vessel (5).

All patent and non-patent references cited in the present applicationare hereby incorporated by reference in their entirety.

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The terms “a”, “an” and “the” as used herein are to be construed tocover both the singular and the plural, unless otherwise indicated orclearly contradicted by context.

1. A bioreactor system for producing a product selected from a cell anda biopolymer expressed by a cell, wherein the bioreactor system is acontinuous production system and wherein the bioreactor systemcomprises: a cell culture vessel (5) comprising a product harvest module(6); optionally, a bleed outlet (9); a medium container (1;2); a waterand/or buffer supply (3); and an inline medium dilution system (4) fordiluting concentrated medium from the medium container, the inlinemedium dilution system having an inlet and an outlet, wherein the inletis in fluid communication with the medium container and the water/buffersupply, and wherein the outlet is in fluid communication with theculture vessel.
 2. The bioreactor system of claim 1, further comprisinga sensor connected to the inline medium dilution system, wherein thesensor is located within or outside the cell culture vessel which sensorcan measure the concentration or the amount of medium or the amount ofselected components or nutrients in the cell culture vessel.
 3. Thebioreactor system of any of claim 1, comprising an impurity filter unit(8).
 4. The bioreactor system of claim 1, wherein the inline mediumdilution system contains conductivity and/or pH process analyzers. 5.The bioreactor system of claim 1, wherein the medium container has avolume of at least 50 L, such as at least 100 L, e.g. at least 250 L. 6.The bioreactor system of claim 1, comprising at least two mediumcontainers, each medium container being in fluid communication with aninlet of the inline medium dilution system.
 7. A method for producing abiopolymer using a bioreactor system according to claim 1, the methodcomprising: (a) fermenting cells expressing the biopolymer in a suitablemedium under suitable conditions to allow expression of the biopolymerby the cells, (b) harvesting the biopolymer by removing mediumcomprising biopolymer and impurities via the product harvest module, and(c) isolating the biopolymer from the harvested medium; wherein, duringfermentation, the method further comprises: adding concentrated mediumfrom a medium container and water or buffer from the water/buffer supplyto an inlet of the inline medium dilution system to result in dilutedmedium, and feeding the diluted medium containing nutrients to theculture vessel through an outlet to replenish nutrients consumed by thecells and to compensate for medium removed for harvesting of thebiopolymer.
 8. The method of claim 7, wherein concentrated medium in themedium container is diluted at least by a factor of at least two withwater or buffer before being fed to the culture vessel, e.g. by a factorof at least three, four or five.
 9. The method according to claim 7,wherein concentrated medium from at least two medium containers is mixedwith water or buffer prior to being fed to the culture vessel.
 10. Themethod of claim 7, wherein the cell density in the cell culture vesselduring the fermentation reaches at least 10 million cells per ml medium,e.g. at least 20 million cells per ml medium, such as at least 30million cells per ml medium, such as at least 40 million cells per mlmedium.
 11. The method according to claim 7, wherein concentrated mediumis mixed with water/buffer and the concentrated medium in the mediumcontainer(s) is kept at a reduced temperature of below room temperature,e.g. 1-10° C., such as about 5° C., wherein the concentrated medium hasbeen pre-heated, or wherein the diluted medium is pre-heated from areduced temperature of below room temperature, e.g. 1-10° C., such asabout 5° C. prior to being added to the culture vessel.
 12. The methodaccording to claim 7, wherein the cells are mammalian cells, e.g.selected from the group consisting of CHO (Chinese hamster ovary) cells,hybridomas, BHK (baby hamster kidney) cells, myeloma cells, HEK-293cells, PER.C6® cells, human lymphoblastoid cells and NSO cells.
 13. Themethod according to claim 7, wherein the biopolymer is a polypeptide orprotein, e.g. an antibody or antibody fragment, a blood clotting factor,a cytokine, an enzyme or a peptide hormone; or a virus particle or partthereof.